Polyclonal-monoclonal ELISA assay for detecting N-terminus pro-BNP

Abstract

A specific and sensitive in vitro ELISA assay and diagnostic test kit is disclosed for determining levels of NT-proBNP protein in a variety of bodily fluids, non-limiting examples of which are blood, serum, plasma, urine and the like.

What is claimed is: 
     
       1. A process for determining concentration of N-terminal pro-brain natriuretic peptide (NT-proBNP) in a bodily fluid sample, comprising:
 (a) providing isolated polyclonal antibodies specific for the sequence of amino acid residues 26-51 of SEQ ID No: 1, wherein the isolated polyclonal antibodies are immobilized on a solid support; 
 (b) providing a bodily fluid sample from a patient, wherein said bodily fluid sample is suspected of containing antigenic fragments of NT-proBNP; 
 (c) contacting said bodily fluid sample to said isolated polyclonal antibodies immobilized on the solid support to form a first mixture wherein said antigenic fragments of NT-proBNP in said bodily fluid sample are captured by said isolated polyclonal antibodies; 
 (d) washing the first mixture, wherein the first mixture comprises said antigenic fragments of NT-proBNP captured by said isolated polyclonal antibodies immobilized on the solid phase, thereby forming a washed first mixture; 
 (e) providing monoclonal antibodies produced by a hybridoma cell line designated as 6G11-F11-D12 and deposited with the American Type Culture Collection (ATCC) as Accession Number PTA-4844, wherein said monoclonal antibodies are specific for the sequence of amino acid residues 1-25 of SEQ ID NO:1; 
 (f) labeling said monoclonal antibodies with a detector, thereby forming labeled monoclonal antibodies; 
 (g) adding the labeled monoclonal antibodies of step (f) to the washed first mixture of step (d) wherein said monoclonal antibodies bind to said antigenic fragments of NT-proBNP captured by the isolated polyclonal antibodies immobilized on the solid phase in step (c), thereby forming a second mixture; 
 (h) washing the second mixture, wherein the second mixture comprises captured antigenic fragments of NT-proBNP bound by the labeled monoclonal antibodies; 
 (i) adding a reporter that reacts with said detector of the labeled monoclonal antibodies to form a reaction product; and 
 (j) measuring said reaction product to determine concentration of NT-proBNP in said sample. 
 
     
     
       2. The process of  claim 1 , further comprising comparing the determined concentration of NT-proBNP in said sample with a selected cut-off concentration. 
     
     
       3. The process of  claim 1 , wherein said bodily fluid sample is plasma. 
     
     
       4. The process of  claim 1 , wherein the detector is biotin. 
     
     
       5. The process of  claim 1 , wherein the reporter is peroxidase-labeled streptavidin. 
     
     
       6. A kit for determining a concentration of N-terminal pro-brain natriurectic peptide (NT-proBNP) in a bodily fluid sample, the kit comprising:
 isolated polyclonal antibodies specific for the sequence of amino acid residues 26-51 of SEQ ID No: 1 that are attached to a solid support; 
 monoclonal antibodies produced by a hybridoma cell line designated as 6G11-F11-D12 and deposited with the American Type Culture Collection (ATCC) as Accession Number PTA-4844, wherein said monoclonal antibodies are specific for the sequence of amino acid residues 1-25 of SEQ ID NO:1, wherein the monoclonal antibodies are labeled with a detector; and 
 a reporter capable of reacting with said detector to form a reaction product. 
 
     
     
       7. The kit of  claim 6 , wherein the solid support attached with isolated polyclonal antibodies is a microtitration strip, and wherein the polyclonal antibodies specific for the sequence of amino acid residues 26-51 of SEQ ID NO: 1 are goat antibodies. 
     
     
       8. The kit of  claim 6 , wherein the detector is biotin. 
     
     
       9. The kit of  claim 8 , wherein the reporter is peroxidase-labeled streptavidin. 
     
     
       10. The kit of  claim 9 , wherein the peroxidase is a horseradish peroxidase. 
     
     
       11. The kit of  claim 6 , further comprising a chromogen solution that comprises 3, 3′, 5, 5′- tetramethylbenzidine (TMB) substrate solution. 
     
     
       12. The kit of  claim 6 , further comprising a wash concentrate that comprises phosphate buffered saline with nonionic detergent. 
     
     
       13. The kit of  claim 6 , further comprising a stopping solution that comprises sulfuric acid.